Albumin is the most abundant plasma protein, generally constituting slightly over one-half of the total protein in mammalian plasma. In the human body, albumin has the important role of regulating the water balance between blood and tissues, and of functioning as a transport molecule for various compounds, such as bilirubin, fatty acids, cortisol, thyroxine and drugs such as sulfonamides and barbiturates, that are only slightly soluble in water. An albumin deficiency in individuals is indicated by an abnormal accumulation of serous fluid, or edema; and the albumin deficiency can restrict the transport of slightly water soluble materials throughout the body. Therefore, it is clinically important to determine whether an individual has a deficiency of serum albumin.
In addition, relatively high concentrations of albumin in the urine of an individual is indicative of a diseased condition. For example, the average normal concentration of protein in urine varies from about 2 mg/dL to about 8 mg/dL, with approximately one-third of the total urinary protein being serum albumin. However, in a majority of diseased states, urinary protein levels increase appreciably, such that albumin accounts for from about 60 percent to about 90 percent of the excreted protein. The presence of an abnormal increased amount of protein in the urine, known as proteinuria, is one of the most significant indicators of renal disease, and can be indicative of various other non-renal related diseases.
Therefore, in order to determine if an individual has an albumin deficiency and/or to determine if an individual excretes an excess amount of albumin, and in order to monitor the course of medical treatment to determine the effectiveness of the treatment, simple, accurate and inexpensive albumin detection assays have been developed. Furthermore, of the several different assay methods developed for the detection and/or measurement of albumin in urine and serum, the methods based on dye binding techniques have proven especially useful because dye binding methods are readily automated and provide reproducible and accurate results.
In general, dye binding techniques utilize pH indicator dyes that are capable of interacting with a protein, such as albumin, and that are capable of changing color upon interaction with a protein absent any change in pH. When a pH indicator dye interacts, or binds, to a protein, the apparent pK.sub.a (acid dissociation constant) of the indicator dye is altered and a color change occurs in the dye, producing the so-called "protein-error" phenomenon. In methods utilizing the dye binding technique, an appropriate buffer maintains the pH indicator dye at a constant pH to prevent a color change in the pH indicator dye due to a substantial shift in pH. Due to the "protein-error" phenomena, upon interaction with the protein, the pH indicator dye undergoes a color transition that is identical to the color change occurring because of a change in the pH. Examples of pH indicator dyes that are used in the wet phase chemistry assay of proteins capable of interacting or binding to protein and exhibiting "protein-error" color changes include methyl orange, bromocresol purple, bromophenol blue, and bromocresol green. Similarly, dry phase chemistry test strips utilize the pH indicator dyes tetrabromophenol blue and tetrachlorophenol-3,4,5,6-tetrabromosulfonephthalein.
Although pH indicator dyes have been used extensively in protein assays, several problems and disadvantages still exist in protein assay methods utilizing indicator dyes. For example, pH indicator dyes are useful only within narrow pH ranges. Outside the useful range of a dye, the dye either fails to change color upon interaction with the protein or the dye changes color prematurely. More importantly, and more difficult to overcome, albumin assays utilizing pH indicator dyes generally are conducted at relatively low pH (e.g., down to a pH of approximately 2 to 3; and normally at a pH of approximately 5 or below) in order to increase the interaction of the indicator dye with albumin and to permit a color transition to occur in the more commonly used dyes, such as bromocresol green and bromocresol purple, as a result of a pKa shift. However, since the acid-base transitions, and therefore the color transitions, of the more commonly used dyes occur at acidic pH values, non-specific interaction of these dyes with protein molecules other than albumin is increased. This potentially undesirable side effect occurs because at acidic pH values most proteins are cationic, or positively-charged, whereas the commonly used dye molecules can exist as negatively-charged anions. Therefore, interaction between the positively-charged cationic protein and negatively-charged anionic dye molecule is promoted. However, at higher pH values, most proteins become neutral or negatively-charged, and the non-specific ionic interactions with the indicator dye therefore are reduced.
Several simple semiquantitative tests and several complex quantitative tests are available for the determination of the total protein content in urine. The majority of these assay methods, with the notable exception of the simple colorimetric reagent test strip, require the precipitation of protein to make quantitative protein determinations. However, the colorimetric reagent test strip utilizes the previously discussed ability of proteins to interact with certain acid-base indicators and to alter the color of the indicator without a change in the pH. For example, when the indicator tetrabromophenol blue is buffered to maintain a constant pH of approximately 3, the indicator imparts a yellow color to solutions that do not contain protein; however, for solutions containing protein, the presence of protein causes the buffered dye to impart either a green color or a blue color to solution, depending upon the concentration of protein in the solution.
Some colorimetric test strips used in protein assays have a single test area consisting of a small square pad of absorbent paper impregnated with a buffered pH indicator dye, such as tetrabromophenol blue. Other colorimetric test strips are multideterminant reagent strips that include one test area for protein assay as described above, and further include several additional test areas on the same strip to permit the simultaneous assay of other urinary constituents. For both types of colorimetric test strips, the assay for protein in urine is performed simply by dipping the colorimetric test strip into a well mixed, uncentrifuged urine sample, then comparing the resulting color of the test area of the test strip to a standardized color chart provided on the colorimetric test strip bottle.
For test strips utilizing tetrabromophenol blue, buffered at pH 3, as the indicator dye, semiquantitative assays for protein can be performed and are reported as negative, trace, or one "plus" to four "plus". A negative reading, or yellow color, indicates that the urine contains no protein, as shown by the lack of color change of the indicator dye. A trace reading detects from about 5 to about 20 mg/dL of protein in the urine. The one "plus" to four "plus" readings, signified by a color change from green through increasingly dark shades of blue, are approximately equivalent to urine protein concentrations of 30, 100, 300, and 1000 mg/dL respectively, and serve as reliable indicators of increasingly severe proteinuria. It should be noted that a highly buffered, alkaline urine can give a false positive assay should the buffer system in the reagent test area be overcome and an actual shift in pH of the buffer occur.
The literature on the technology of dye binding to proteins is extensive and shows that protein sensitive dyes generally function at a fairly narrow pH range in the acidic region. Accordingly, Sanford, in U.S. Pat. No. 4,568,647, is the only known reference relating to a protein indicator dye that functions in the alkaline pH region.
Sanford discloses a method of assaying aqueous liquids for albumin, in a pH range of about 5 to about 11, utilizing a dye having a heterocyclic ring or a carbocyclic ring terminating each end of either a vinyl group or a conjugated polyene chain of up to seven carbon atoms. The dyes disclosed by Sanford preferentially bind to albumin over other proteins, and upon binding to albumin, the dyes exhibit a detectable shift in spectral absorption. The amount of albumin in the aqueous test sample can be quantitatively determined by measuring and correlating energy absorption of the aqueous test sample at the absorption maximum to the absorptions of solutions of known albumin concentration at the absorption maximum.
In contrast to the prior art and in contrast to the presently available commercial test strips, the indicator dyes useful in the method of the present invention can interact with albumin and as a result undergo a detectable color transition in a pH range that approximates the natural pH of urine and serum. Therefore, in accordance with an important feature of the present invention, extensive buffering of the indicator dye at an acidic pH is not required. As a result, the contamination of other assay pads, such as the urine pH assay pad, on a multiple pad test strip by the acidic components of the albumin assay pad is reduced or eliminated, thereby precluding the albumin assay from interfering with the simultaneous assay of other urine constituents.
Therefore, to avoid the potential contamination and resulting interferences associated with assays buffered in the acid pH region, it would be extremely advantageous to provide a method and composition for the assay of albumin in urine or serum at an essentially neutral to an alkaline pH. Likewise, it would be advantageous if the albumin assay method could be utilized in a dip-and-read format for the easy and economical, qualitative and/or semiquantitative determination of albumin in urine or serum.
Furthermore, any method for the assay of albumin in urine or serum must give accurate, trustworthy and reproducible results by utilizing a composition that undergoes a color transition as a result of an interaction with albumin, and not as a result of a competing chemical or physical interaction, such as preferential binding to proteins other than albumin. In addition, the method and composition for the albumin assay should be suitable for use both in wet assays and in dry reagent strips for the rapid, economical and accurate determination of albumin in urine or serum. Also, the method and composition utilized in the assay for albumin must not adversely affect or interfere with the other test reagent pads that are present on multiple test pad strips.
Prior to the present invention, no known composition or method of assaying urine or serum for albumin included a trisubstituted methane and/or a substituted phenolphthalein-type compound, that is capable of interacting with albumin and that is capable of undergoing a color transition at an essentially neutral to alkaline pH, as the indicator dye. Hence, in accordance with the method and composition of the present invention, new and unexpected results are achieved in the dry reagent strip assay and the wet assay of urine and serum for albumin by utilizing a trisubstituted methane indicator dye and/or a substituted phenolphthalein-type compound indicator dye buffered at an essentially neutral to an alkaline pH.